TP53 and KMT2D mutations associated with worse prognosis in peripheral T‐cell lymphomas

Abstract There are limited studies on mutation profiling for Peripheral T‐cell lymphomas (PTCL) in the Chinese population. We retrospectively analyzed the clinical and genetic landscape of 66 newly diagnosed Chinese patients. Targeted next‐generation sequencing (NGS) was performed for tissues from these patients. At least one mutation was detected in 60 (90.9%) patients, with a median number of 3 (0–7) mutations, and 32 (48.5%) cases detected with more than 4 mutations. The genes with higher mutation frequencies were TET2, RHOA, DNMT3A, IDH2, TP53, STAT3, and KMT2D respectively. When mutant genes are classified by functional group, the most prevalent mutations are related to epigenetics and signal transduction. IPI ≥2, PIT ≥2, and failure to achieve partial remission (PR) were factors for inferior progression‐free survival (PFS) and overall survival (OS). Multivariate analysis showed TP53 was an adverse factor for PFS (HR, 3.523; 95% CI, 1.262–9.835; p = 0.016), and KMT2D was an adverse factor for OS (HR, 10.097; 95% CI, 1.000–101.953; p = 0.048). Mutation profiling could help differentiate distinct types of PTCL and serve as a useful tool for determining treatment options and prognoses.


| BACKGROUND
PTCL are a heterogeneous group of proliferative disorders of mature lymphocytes comprising more than 30 distinct subtypes. 1Although they are all derived from either postthymic T cells or NK cells, the disease biology and genomic features of the different subtypes vary considerably.The pathogenesis of PTCL is a complex process involving dysregulation of the TCR pathway, viral and chronic inflammation-driven chromosomal translocations, insertions, deletions, and mutations. 2In addition, a wide range of epigenetic abnormalities plays a critical role in tumor chemoresistance and disease progression (PD). 3Except for Anaplastic Lymphoma Kinase (ALK) positive anaplastic large cell lymphoma (ALCL), patients with PTCL generally have a poor prognosis.CHOP (Cyclophosphamide, doxorubicin, vincristine, and prednisolone)-based chemotherapy remains the "standard" treatment.8][9] With the application of next-generation sequencing (NGS) technology in recent years, genomics research has been widely carried out in PTCL, mainly including whole genome sequencing (WGS), exome sequencing, transcriptome sequencing analysis, genomic polymorphism analysis based on gene chip technology, and epigenetic studies. 10,11However, there has been very little study on PTCL that covers the primary signaling pathways involved, gene expression profiling (GEP) characterization, and the prognosis of Chinese PTCL patients.By performing targeted sequencing of 103 lymphoma-associated genes in 66 newly diagnosed PTCL patients and analyzing the correlation between highfrequency gene mutations and clinical characteristics, we explored the mutation profiling and GEP characteristics of PTCL patients in this study.

| Data collection
We included 66 cases of PTCL admitted to the Bone Marrow Transplantation Center of the First Affiliated Hospital of Zhejiang University School of Medicine from July 2020 to November 2023 in this study.All cases were diagnosed as PTCL according to hematolymphoid neoplasms: the 5th edition of the WHO classification (WHO-HAEM5).Inclusion criteria included biopsy specimens with a preliminary diagnosis and histologically confirmed PTCL, including subtypes such as extranodal natural killer (NK) TCL nasal type, Peripheral T-cell Lymphoma, Not Otherwise Specified (PTCL-NOS), Angioimmunoblastic T-cell Lymphoma (AITL), and ALK-ALCL.Immunohistochemistry mainly included CD20, CD3, CD10, BCL-6, Ki-67, CD5, CD30, CD2, CD4, CD8, CD7, CD56, CD21, CD23, EBER-ISH, TCRβ, TCRδ, PD1/ CD279, ALK, and TP63.Cell surface markers for flow cytometry included: CD45, CD3, CD5, CD19, CD10, CD20, CD30, CD4, CD8, CD7, CD2, TCRαβ, and TCRγδ.All pathological tissue slides were diagnosed and reviewed by two experienced pathologists.All cases were treated with first-line therapy and were evaluated with PET-CT or CT.Patients who did not achieve PR were switched to second-line therapy or entered clinical trials.Exclusion criteria: patients without complete clinical data or lost to follow-up, patients who had received radiotherapy or chemotherapy before enrollment, and patients with comorbidities of other hematological neoplasms and malignant consumptive disease.Clinical baseline data were collected from all patients at the time of initial diagnosis, including gender, age, lumbar disc herniation (LDH), Epstein-Barr virus (EBV)-DNA, Ann Arbor stage, Eastern Cooperative Oncology Group Performance Status (ECOG PS), bone marrow biopsy, B symptoms, treatments, efficiency (complete remission [CR] or PR, stable disease [SD], or PD) and outcomes.PFS was calculated using the date of PD or death, with the latter being used to compute OS.The International Prognostic Index (IPI) and the Prognostic Index for TCL (PIT) were computed using the International Prognostic Factors in Non-Hodgkin's Lymphoma Project system and the Intergruppo Italiano Linfomi system, respectively.All patients gave informed consent, and the study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine before implementation.Furthermore, it was carried out in compliance with the Helsinki Declaration.

| NGS sequencing
Formalin-fixed, paraffin-embedded tissue slices (FFPE) were used to extract genomic DNA at diagnosis.The samples were analyzed by hybridization-capture sequencing using Illumina High-Throughput Sequencing Platform technology, with an average sequencing depth of 16,066X and a coverage rate of >90%.The gene Pannel included exons, fusion-associated intronic regions, and variable shear regions of 103 genes related to lymphoma, totaling about 366,802 loci.DNA sequencing was performed in all patients but RNA sequencing was done in only 30 patients, and no clinically significant fusion or mutant genes were detected.Therefore, only the results of DNA sequencing were shown in the study.

| Follow-up
All patients were followed up until November 30, 2023, and follow-up information was obtained by querying inpatient medical records, outpatient medical records, and telephone follow-ups.PFS was defined as the period from the beginning of diagnosis of PTCL to PD or the time to the final follow-up.OS was defined as the time from the diagnosis of PTCL until the patient's death or the last follow-up.

| Statistical analysis
SPSS 26.0 was used for data analysis, and R-3.6.1 statistical software was used to draw heat maps.A comparison of baseline clinical characteristics of the samples was performed using the chi-square test to calculate p values.OS and PFS were analyzed by the Kaplan-Meier curve analysis with the log-rank test.Multivariate prognostic correlation analysis was performed using the Cox proportional risk regression model.p < 0.05 was considered a statistically significant difference.

| DISCUSSION
The classification of PTCL is complex, with more than 30 subtypes in the latest WHO-HAEM5.Although pathology is the gold standard for diagnosis, it is highly susceptible to misdiagnosis due to the high degree of heterogeneity, and patients often require multiple repeat tests or consultation with experienced pathologists, resulting in a long diagnostic cycle. 12][15] IPI is more commonly used in patients with NHL.In patients with PTCL, IPI showed some prognostic value for OS and PFS, but some low-risk patients with IPI also had a poorer prognosis.PIT was similarly deficient.None of the available prognostic models provide much guidance for treatment decisions.Mutation profiles and GEP may help to distinguish different types of PTCL and identify high-risk patients.In our study, TP53 was the only independent poor prognostic factor associated with PFS.][18] Genomic instability caused by TP53 alterations induces cancer chemoresistance and progression. 19,202][23] However, the prognosis of these patients remains unfavorable even after high-dose therapy.In our study, patients with TP53 mutations were all male.Five of them were older than 60 years old, six had B symptoms, eight were in stage III or IV, and four cases died.Three out of nine cases received HSCT and all survived.Six were combined with DNMT3A, followed by TET2 (n = 4), STAT3 (n = 3), RHOA (n = 2), IDH2 (n = 1), FAS (n = 1), KRAS (n = 1), MTOR (n = 1), and KMT2D (n = 1).Notably, although TP53 mutations are the inferior factor for PFS, but there was no statistical difference on OS.Regarding  the impact of TP53 mutations on the prognosis of PTCL, our findings were consistent with the study by William T. Johnson et al.They hypothesized that novel clinical trials of noncytotoxic therapies, or performing curative allo-HSCT allowed patients with TP53 mutations to achieve OS similar to that of TP53-negative patients. 24MT2D mutation was an independent poor prognostic factor associated with OS.Patients with KMT2D mutation were all classified as intermediate-to highrisk group.40% (2/5) patients died of PD.Four patients were treated with conventional CHOP chemotherapy in combination with oral Chidamide, three of them were evaluated as PR.The other case was treated with CHOP combined with Azacitidine, and the efficacy was assessed as SD.No patients were detected with combined TET2, RHOA, IDH2, MTOR, or KRAS mutations.Combined FAS mutations were found in 2 cases, and TP53, STAT3, and FAT1 in 1 case, separately.KMT2D co-mutated with high-frequency genes less often and was more likely to present as a single mutation.KMT2D is related to histone methylation.Also known as MLL2 or MLL4, it encodes histone-lysine N-methyltransferase, which methylates histone H3 to activate transcription.A study that included 123 EBV-associated T or NK cell lymphoproliferative disorders (EBV + T/NK-LPD) patients found that KMT2D mutations were detected in 12 of 87 nasal-ENKTL.It was also the gene with the highest frequency (30.6%, 11/36) in the remaining 36 patients with ENKTL.Survival analysis suggested that KMT2D mutations in both nasal and extranasal ENKTL subgroups suggested worse OS (p = 0.002). 25KMT2D mutations likewise suggest a poor prognosis in B-cell lymphoma. 26The FIL-MCL0208 phase 3 trial showed that mutations of KMT2D and disruption of TP53 by deletion or mutation are linked to an elevated risk of progression and mortality.To our knowledge, the adverse effects of KMT2D mutations on survival in PTCL have not been documented to date.There could be two primary explanations for why KMT2D mutations did not affect survival in earlier research.First, not all KMT2D mutations fit the definition of "true" mutations because some of them are missense sequence variants that have not been reported as somatic mutations.Second, the insufficient number of NGS-tested cases in earlier research could have resulted in an underestimation of "true" mutations.
There are very few studies on mutated genes influencing the prognosis of PTCL.Apart from the mutations in TP53 and KMT2D that we have detected, there have been reports of other genetic markers that are associated with a poor prognosis.5][26][27][28][29][30] In our study, the top five genes in PTCL were TET2 (53%), RHOA (33%), DNMT3A (26%), IDH2 (18%), and TP53 (14%).There were 63 kinds of amino acid alterations corresponding to the TET2 mutations, further validating the heterogeneity of PTCL.The genetic profile of these mutations suggests that PTCL is a complex spectrum of mutations that integrally cause events in a multistep, multi-target oncogenic process.Analyzing the altered molecular mechanisms can provide molecular pathways and gene targets for studying the mechanisms of PTCL.We will expand the number of specimens and verify the role of these genes in PTCL at the protein level and other aspects to provide a theoretical basis for diagnosis and assessment of prognosis.In addition, given the adverse effects of TP53 and KMT2D on patient prognosis, the subsequent study at our center is to expand the size of cases and establish an independent prospective cohort for validation.We plan to incorporate the two risk factors, TP53 and KMT2D, to develop a new prognostic model to identify patients with truly high-risk PTCL, and to explore new treatment strategies for these patients.To increase the number of patients with PTCL by enhancing WES, targeted sequencing, and RNA-seq assays, we plan to set up multicenter research.A complete mutant genomic map of this population will be presented by the application of multi-omics research.Create molecular stratification according to treatment response, physiological alterations, and genetic characteristics.In addition, to explore whether microenvironmental stratification can be established based on gene expression of infiltrating immune cells and stromal components.
This retrospective study, which primarily included patients from cities in southern China and where all participants underwent NGS testing before treatment, is the largest cohort of PTCL in the Chinese population reported to date.Consistent with previous studies, we confirmed that PTCL was predominantly in older male patients.No trend toward the beneficial effects of adding Azacitidine was observed in PFS and OS.Although most PTCL patients are sensitive to chemotherapy, remission durations are usually short, and recurrent disease relapses lead to poor OS.In this study, TP53-mutant patients who received HSCT showed superior outcome benefit compared to patients who did not receive HSCT.The results of our study further suggest that HSCT is still recommended for patients with high-risk prognostic factors to achieve better F I G U R E 4 PIT≥2, failure to achieve PR, TET2 mutation, and TP53 mutation suggest worse PFS (p = 0.270, p < 0.001, p = 0.256, and p = 0.075, respectively).Although this study provides Chinese data on PTCL, there are some limitations.The patients enrolled were mainly from southern cities and the follow-up period was still short.AITL and PTCL-NOS constituted the majority of the histologic subtypes, with fewer types such as ENKTCL and ALCL.Regrettably, we did not perform the TBX21 assay for PTCL-NOS in this study.Therefore, it was not possible to further analyze the subtypes of GATA3, TBX21, and TFH.Furthermore, due to the retrospective character of the study, and the patients were treated heterogeneously, the possibility of unidentified bias cannot be ruled out, and more rigorous matching studies and regression analyses are needed to exclude confounding by baseline imbalances between groups.

| CONCLUSION
PTCL is a rare and highly heterogeneous group of hematologic malignancies that are aggressive and characterized by a poor prognosis.There are differences in the prognosis and gene distribution between subtypes.Based on clinical, phenotypic, and genetic characteristics, future research ought to look into the molecular pathophysiology of PTCL as well as the influence of the tumor microenvironment on treatment and drug resistance.Our study integrates NGS of 66 patients with PTCL to depict a comprehensive GEP of PTCL in Chinese patients.The TP53 mutation defines a high-risk group with minimal response to standard or intensified treatment.Our research provides critical evidence that patients with KMT2D and TP53 mutations indicate poor outcomes.Subsequent research endeavors aim to construct molecular stratification according to genetic attributes, biological modifications, and responsiveness to treatment.To identify genuinely high-risk patients, a novel predictive model that incorporates TP53 and KMT2D will also validated by an independent prospective cohort.According to our research, genes with higher mutation frequencies are linked to epigenetic modifications.Therefore, the use of demethylating agents in conjunction with combination immunotherapy or lower dosages of conventional chemotherapy may be taken into consideration to lessen the toxicities associated with chemotherapy in elderly, frail patients.The impact of KMT2D on the prognosis of PTCL was not addressed in earlier research.We hypothesize that this could be attributed to limited cases and insufficient genetic testing, which has left us with little knowledge of this gene.
According to our observations in southern Chinese cities, mutation profiling and GEP may help differentiate distinct types of PTCL and serve as a useful tool for determining treatment options and prognoses.

F I G U R E 1
An oncoplot of all 66 patients' genetic mutations.Every column denotes an independent patient.The number of alterations detected in each sample is shown on the top row.The type of mutation and diagnosis are represented by each row.Wild type is indicated by gray tiles.Mutations for that particular gene are represented by percent frequencies in the far-right column.
Baseline clinical characteristics of enrolled patients.
T A B L E 1 The gene mutation spectrum.
Multivariate Cox regression model for PFS and OS.Summary of mutational landscape and survival outcome of PTCL. .However, in China, the proportion of patients undergoing HSCT after first-line treatment is very low due to the limitations of advanced age, education, occupation distribution, and economic factors.
T A B L E 5survival